Abstract: The mitotic spindle uses multiple force-generators and regulatory molecules to assemble itself and segregate sister chromatids. The broad aim of the work described here is to provide a comprehensive molecular and quantitative explanation of how ensembles of mitotic proteins cooperate to produce the dynamic and mechanical properties of the spindle during its assembly, maintenance and elongation. The conceptual framework underlying the proposal is that mitotic movements depend on transitions between steady state structures. Steady state structures e.g. metaphase spindles at constant length are maintained by a balance of forces, generated by multiple force generators. Mitotic movements (e.g. spindle pole separation during spindle assembly and anaphase spindle elongation) occur when the balance is tipped. The specific aims are 1. To test and elucidate the molecular mechanisms of our model for early spindle assembly which depends on a force-balance generated by MT-based motors, cortical motors and nuclear elasticity 2. To test the hypothesis that bipolar kinesin-5 motors crosslink MTs and drive a sliding filament mechanism that is antagonized by kinesin-14 to maintain the prometaphase spindle, using motility assays and cryo-electron microscopy. 3. To test and elucidate the molecular details of our model for anaphase B in which a kinesin-5-driven interpolar (ip) MT sliding filament mechanism generates motile force and poleward MT flux acts as an on-off switch. 4. To begin a high-throughput, system-level analysis of the mitotic network. Our experiments will utilize dynamic time-lapse imaging of control versus inhibitor-injected and mitotic mutant Drosophila embryos;high-throughput fluorescence microscopy of cultured S2 cells following RNAi depletion of mitotic proteins;and biochemical analysis of mitotic proteins prepared from native or baculovirus-infected insect cells. Complementary quantitative modeling will exploit systems of force-balance and rate equations to explain the dynamics of specific phases of mitosis in terms of the underlying molecules. We aim to understand how the mitotic spindle works as a machine and thus to provide insights into how defects in its action can give rise to genomic instability, cancer and birth defects.